Reactive oxygen species (ROS), endogenously generated as respiration by-products and exogenously by radiation and other genotoxins, induce a variety of base damage and DNA strand breaks in cell genomes, which are mostly repaired via the base excision repair (BER) pathway, initiated with excision of base lesions by DNA glycosylases. Accumulation of oxidized base damage without a strong phenotype in mice lacking OGG1 and NTH1, the major mammalian glycosylases, suggested that additional glycosylases repair active genomic sequences. In our previous project period we characterized human glycosylases (NEIL1 and NEIL2) with overlapping substrate preferences as OGG1 and NTH1. However, only the NEILs excise lesions from single-stranded or bubble DNAs. Stable interaction of NEIL2 with RNA polymerase II, and of NEIL1 with the sliding clamp PCNA, and other DNA replication-associated proteins led us to hypothesize a preferential role for NEIL1 and -2 in replication (RAR)- or transcription-associated repair, respectively. We further postulate that NEIL1 preferentially repairs base damage in leading or lagging template strand (preplicative repair), and also removes incorporated base lesion in the nascent DNA strand (post-replicative repair). Formation of multiprotein complexes in response to radiation and oxidative stress, which may also be affected by covalent modification of component proteins, led us to hypothesize that distinct repair complexes are assembled for repairing endogenous vs. radiation/ROS-induced damage, and in G1 vs. S-phase cells, by utilizing distinct DNA polymerases and other proteins including RPA, FEN1 (activated by PCNA) and Werner or Bloom protein with DNA helicase activity. In this competing renewal project we will examine various facets of this hypothesis by testing whether (1) NEIL1 forms distinct repair complexes in G1 vs. S-phase cells, and in response to oxidative stress;(2) NEIL1 preferentially repairs base damage in both template and nascent DNA during replication by interacting with the replicating complex;(3) damage in replicating plasmid is preferentially repaired in vivo relative to nonreplicating plasmid;and (4) NEIL1-induced cleavage at the damage site of the replicating DNA strand leads to double-strand breaks. Health Relevance: Successful completion of these studies will not only establish the novel concept of RAR, but will also illuminate various interactions among repair proteins distinct complexes. Such basic understanding of interaction interfaces in repair complexes could be exploited in developing therapeutic intervention strategies for sensitizing tumor cells or protecting healthy cells during chemo/radiation therapy.